Novel Biomarkers of 3-Chloro-1,2-propanediol ... - ACS Publications

Feb 17, 2010 - Chinese Center for Disease Control and PreVention, and Department of Health Statistics, Public Health College,. Harbin Medical UniVersi...
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Chem. Res. Toxicol. 2010, 23, 1012–1017

Novel Biomarkers of 3-Chloro-1,2-propanediol Exposure by Ultra Performance Liquid Chromatography/Mass Spectrometry Based Metabonomic Analysis of Rat Urine Ying Li,† Shuang Liu,† Cheng Wang,‡ Kang Li,§ Yu-Juan Shan,† Xi-Jun Wang,| and Chang-Hao Sun*,† Department of Nutrition and Food Hygiene, Public Health College, Center for Endemic Disease Control, Chinese Center for Disease Control and PreVention, and Department of Health Statistics, Public Health College, Harbin Medical UniVersity, Harbin, China 150081, and Pharmacy Department, Heilongjiang UniVersity of Chinese Medicine, Harbin, China 150040 ReceiVed NoVember 1, 2009

To select early, sensitive biomarkers of 3-chloro-1,2-propanediol (3-MCPD) exposure, a single dose of 30 mg/kg/day 3-MCPD was administered to male Wistar rats for 40 days. Significant elevations of serum creatinine and blood urea nitrogen concentrations were observed on day 40, and urine N-acetylβ-D-glucosaminidase and β-galactosidase (β-Gal) activities were observed on day 20. Slight renal tubule hydropic degeneration and spermatozoa decreases were observed on day 10. The endogenous metabolite profile of rat urine was investigated by ultra performance liquid chromatography/mass spectrometry with electrospray ionization (ESI). Principal component analysis and partial least-squares enabled clusters to be visualized, with a trend of clustering on day 10 in ESI- and the greatest differences on days 30 and 40. Galactosylglycerol, a marker contributing to the clusters, which had earlier variations than conventional biomarkers and the most significant elevations as compared to other novel biomarkers, was first considered to be an early, sensitive biomarker in evaluating the effect of 3-MCPD exposure. The identification of galactosylglycerol was carried out by β-Gal catalysis, and the possible mechanism of urine galactosylglycerol variation was elucidated. Introduction The traditional way of treating protein-rich extracts from soya beans or other vegetable sources with concentrated hydrochloric acid at high temperature leads to the formation of significant amounts of 3-chloro-1,2-propanediol (3-MCPD) (1). Although manufacturing techniques have been improved, a certain amount of 3-MCPD still occurs as a contaminant in foods and food ingredients, especially acid-hydrolyzed vegetable protein and soya sauce, which may be taken daily by Asian consumers as well as Europeans and Americans. 3-MCPD is capable of crossing the blood-testis barrier and the blood-brain barrier and is distributed widely in the body fluids, which results in general toxicity such as nephrotoxicity, reproductive toxicity, genotoxicity, carcinogenicity, and neurotoxicity (2-5). Daily intake of any reasonable amount of 3-MCPD does not cause acute toxic effects. However, the long-term consumption of 3-MCPD from foods is probably able to produce accumulated toxic effects, to which we should pay special attention. The longterm accumulated 3-MCPD toxicity is clearly harmful to humans; therefore, health evaluation and early injury detection are necessary, and selection of appropriate biomarkers that can accomplish such a task is important. * To whom correspondence should be addressed. Tel: +86-451-87502801. Fax: +86-451-8750-2885. E-mail: [email protected]. † Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University. ‡ Center for Endemic Disease Control, Chinese Center for Disease Control and Prevention, Harbin Medical University. § Department of Health Statistics, Public Health College, Harbin Medical University. | Heilongjiang University of Chinese Medicine.

The Joint FAO/WHO Expert Committee on Food Additives has chosen renal tubule hyperplasia as the most sensitive end point, which has been seen in a long-term study of 3-MCPD toxicity and carcinogenicity in rats (6). Li et al. have studied the toxic effects of 3-MCPD on rats and considered urine N-acetyl-β-D-glucosaminidase (NAG) activity to be a biomarker for assessing the effect of 3-MCPD (7). However, renal tubule hyperplasia is not convenient for human monitoring, and whether NAG is sufficiently sensitive for health evaluation and early injury detection remains unknown. The selection of some other biomarkers that are more sensitive for assessing the longterm health effects of low-dose 3-MCPD exposure and that are more convenient for sample collection and human monitoring is needed. Metabonomics is a sensitive and unbiased analysis method that is aimed at all metabolites in biological samples (8). It has enormous potential to identify novel biomarkers, especially in toxicology, and early studies have been conducted in the field of renal (9-12) and hepatic toxic biomarkers (13-16). Recently, ultra performance liquid chromatography/mass spectrometry (UPLC/MS) in metabonomic studies has been applied widely because of its high sensitivity and reproducibility (17). In addition, urinary biomarkers derived from metabonomic analysis can be detected easily and are convenient for human monitoring. Here, we used UPLC/MS to investigate, possibly for the first time, the metabolic changes caused by 3-MCPD in rat urine and identified a sensitive biomarker in the early period, as compared with histopathology and conventional biomarkers. Our results showed that galactosylglycerol was a novel biomarker for 3-MCPD exposure monitoring, with a high contribution ratio and early, significant variation.

10.1021/tx900400p  2010 American Chemical Society Published on Web 02/17/2010

NoVel Biomarkers of 3-Chloro-1,2-propanediol Exposure

Experimental Procedures Chemicals and Reagents. Acetonitrile (chromatographic grade) was purchased from Tedia (Fairfield, OH). Formic acid (analytical grade) was purchased from Beijing Reagent Co. (Beijing, China). Deionized water was purified by the Milli-Q system (Millipore, Billerica, MA). Leucine enkephalin was purchased from SigmaAldrich (St. Louis, MO). 3-MCPD, with a chemical specification of 99.67%, was purchased from Shenyang Gold Jyouki Chemical Co. (Shenyang, China). The assay kits for NAG, serum creatinine, and blood urea nitrogen (BUN) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The assay kit for β-galactosidase (β-Gal) was purchased from 3 V Co. (Shandong, China), and purified β-Gal was purchased from Worthington Biochemical Corp. (Lakewood, NJ). Animal Handling. Male Wistar rats (220 ( 20 g) were purchased from Basic Medical College of Jilin University (Changchun, China). All rats were allowed to acclimatize in communal plastic cages for 14 days and then in metabolism cages for 3 days before treatment. Temperature and humidity were regulated at 22 ( 2 °C and 45 ( 15%, respectively. A cycle of 12 h light/12 h dark was established. Food and drinking water were provided ad libitum. 3-MCPD was diluted by distilled water to 3‰ v/v. Rats received either 3-MCPD (30 mg/kg/day, n ) 10) or distilled water (10 mL/kg/day, n ) 10) by oral gavage for 40 days. The whole study was divided into five time points, including -1 (the day before treatment), 10, 20, 30, and 40 days. Body weights were measured at each time point. All experimental procedures were conducted in conformity with the institutional guidelines for the care and use of laboratory animals of Harbin Medical University (Heilongjiang, China) and conformed to the National Research Council’s Guide for the Care and Use of Laboratory Animals. Sample Collection. At each time point, urine samples of the two groups (n ) 100) were collected in metabolism cages over 24 h to avoid the effects of diurnal variation on urine metabolite profiles (18, 19), centrifuged at 14000 rpm for 10 min to remove particle contaminants, and stored at -80 °C until analysis. Urine samples collected at five time points were also analyzed for NAG and β-Gal activity by a colorimetric method. Blood samples were collected at 30 and 40 days to test for serum creatinine (Jaffe reaction) and BUN (Fearon reaction). All analysis was performed on a spectrophotometer, except for β-Gal, which was analyzed by automated biochemical analyzer. Histopathology. Rats were sacrificed by exsanguination (n ) 6) from the abdominal aorta under 2% pentobarbital sodium anesthesia at 10, 20, and 30 days, while the remaining 10 rats in the 3-MCPD-treated group and 10 rats in the control group were sacrificed on day 40. Testes and kidneys were excised, dissected free of connective tissue and fat, and weighed. Following fixation in buffered 10% formalin and processing in wax blocks, serial transverse sections were prepared. For each organ, individual sections were stained with hematoxylin and eosin (HE) and examined by light microscopy. UPLC/MS Analysis. UPLC/MS analysis was carried out using a Waters ACQUITY UPLC system (Waters Corp., Milford, MA) coupled to a Waters Micromass Q-tof (Quatropde-Time of Flight) micro Mass Spectrometer (Manchester, United Kingdom) with electrospray ionization (ESI) in positive and negative modes. Urine samples were centrifuged at 14000 rpm for 10 min, and the supernatant was transferred into an autosampler vial. A 3 µL aliquot of supernatant was injected into an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm; Waters Corp.). The flow rate of the mobile phase was 400 µL/min (back pressure, -11000 psi). The column was eluted with a linear gradient, where A was 0.1% formic acid in water and B was 0.1% formic acid in acetonitrile. The initial composition of buffer B was 2% and increased to 32% in 6 min, 62% in 1.5 min, 67% in 1.5 min, and to 98% in a further 2 min, followed by re-equilibration to the initial conditions in 3.0 min. Each run time was 20 min. For MS analysis, the source temperature was set at 110 °C with a cone gas flow of 100 L/h. A desolvation gas temperature of 300

Chem. Res. Toxicol., Vol. 23, No. 6, 2010 1013 °C and a desolvation gas flow of 600 L/h were employed. The capillary voltage was set at 3.2 kV in positive ion mode and 2.8 kV in negative ion mode, and the cone voltage was set to 35 V. All of the analyses were acquired by using the lock spray to ensure accuracy and reproducibility. A lock mass of leucine enkephalin for positive ion mode ([M + H]+ ) 556.2771) and negative ion mode ([M + H]- ) 554.2615) was employed via a lock spray interface. The lock spray frequency was set at 0.48 s, and the lock mass data were averaged over 10 scans for correction. The MS data were collected in full scan mode from m/z 100 to 1000 from 0 to 16 min in positive and negative ion modes. Data Analysis. Statistical analysis was performed by SPSS (version 13.01S; Beijing Stats Data Mining Co. Ltd., China). Differences between groups were analyzed by one-way analysis of variance (ANOVA), repeated measures ANOVA, or Student’s t test. P < 0.05 was considered to be significant. The UPLC/Q-TOF/MS data were analyzed using the MarkerLynx Application Manager (Waters Corp.). The mass window was set at 0.02 Da, the noise elimination level was set at 10.00, the RT tolerance was set at 0.01 min, and the RT window was set at 0.2 min. The resulting 3D matrix that contained arbitrarily assigned peak indexes (retention time-m/z pairs), sample names (observations), and normalized peak area was exported to SAS software 9.1.3 for multivariate statistical analysis using principal component analysis (PCA) and partial least-squares (PLS), which were used to obtain the biomarkers. The UPLC/MS/MS product ion spectrum of biomarkers was matched with the structure message of metabolites (with the same m/z as our biomarkers) obtained from the Human Metabolome Database (HMDB) or Chemspider with Mass Fragment software (Waters Corp.).

Results Clinical Chemistry and Histopathology. 3-MCPD-treated rats had lower body weight gain at 40 days as compared to the control group. Urinalysis showed significant increases in urinary NAG activity in 3-MCPD-treated rats from day 20. BUN and serum creatinine values were similar for controls and 3-MCPDtreated rats on day 30, and both were increased significantly on day 40 (Figure 1 in the Supporting Information). The mean testes-body ratio for the 3-MCPD-treated group was 0.689 (SD ) 0.134), a significant decrease as compared to the control group (mean value, 0.947; SD ) 0.225; P < 0.05). The mean kidneys-body ratio was 0.886 (SD ) 0.081) for the 3-MCPD-treated group, whereas the control group had a value of 0.643 (SD ) 0.142, P < 0.01). A mild to severe decrease in the number of spermatozoa and sustentacular cells in the seminiferous tubules was observed from day 10 to 40. Minimal to severe focal tubule hydropic degeneration was observed in kidneys from day 10 to 40, with an increasing number of foci. Tubular cell hyperplasia was observed in one of the 3-MCPD-treated rats on day 40 (Figure 2 in the Supporting Information and Figure 1). UPLC/MS Fingerprinting and Multivariate Analysis. All of the urine samples collected in this study were analyzed by UPLC/MS with positive and negative ESI. Pattern recognition via PLS and PCA was performed on positive and negative ESI data. The PLS score plots revealed the clustering on day 20 in positive ion mode and on day 10 in negative ion mode (Figure 3 in the Supporting Information). The mean PCA score plots showed that the 3-MCPD-treated group deviated from the control group from day 30 and had the most significant deviation on day 40 (Figure 2). Analysis of these data using PLS and PCA revealed that dosing with 3-MCPD was associated with elevation of a number of compounds in positive and negative ESI modes. The principal ions that increased in positive ion mode after dosing were those

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Figure 1. Photomicrographs of kidney before and after administration. (A) In control rats, no abnormalities were noted; HE stain, magnification, 200×. (B-E) 3-MCPD-treated rats on days 10, 20, 30, and 40, respectively. Aggravated hydropic degeneration was observed; HE stain, magnification, 200×. (F) 3-MCPD-treated rat on day 40, showing tubular cell hyperplasia; HE stain, magnification, 400×.

for m/z 114.0054, 154.9782, 156.0341, 181.0012, 182.9626, 201.9092, 232.1309, 271.0467, 287.1031, 289.1088, 297.1131, 298.1070, 310.1029, 655.2521, 655.3010, and 656.3128, which remain unidentified. The principal ion that increased in negative ion mode after dosing was at m/z 253.0847, which eluted at 4.49 min and was identified as galactosylglycerol (calculated m/z 253.0923; mass error, 30 ppm), which increased significantly on day 10 and kept increasing from then onwards. The UPLC/MS/MS product ion spectrum of galactosylglycerol is shown in Figure 3C. As the authentic standards of these postulated identities were unavailable, it was difficult to make an unequivocal structure determination. Additional ions at m/z 190.8727, 194.9881, 253.9857, 254.9907, and 282.9889 also increased after dosing, but remain unidentified. The variation of these compounds was reproducible in the duplicate experiment (the same biomarkers were obtained).

According to the biological functions and metabolic pathways of galactosylglycerol, the mechanism of galactosylglycerol elevation in 3-MCPD-treated rats was probably associated with β-Gal leakage, which might have been due to 3-MCPD nephrotoxicity and reproductive toxicity. The test of urine β-Gal activity revealed that 3-MCPD-treated rats had significant elevation from day 20 as compared to controls (Figure 3B). For galactosylglycerol identification, 50-100 µL of purified β-Gal was added to seven urine samples with a volume of 500 µL. The whole reaction was carried out at an optimal pH of 6-8 and temperature of 25 °C and lasted for 5-10 min. The control group used the same seven urine samples without β-Gal. All urine samples were immediately analyzed by UPLC/MS with negative ESI before and after the reaction. The relative content of the compound with a molecular weight of 253.0847 and retention time of 4.49 min had a significant reduction after

NoVel Biomarkers of 3-Chloro-1,2-propanediol Exposure

Figure 2. Combined mean UPLC/MS data. (A) Positive ESI trajectory plots for control (rhombus, broken line) and 3-MCPD-treated (circle, solid line) rats, at five time points. (B) Negative ESI trajectory plots for control (rhombus, broken line) and 3-MCPD-treated (circle, solid line) rats, at five time points.

enzyme reaction in the β-Gal group, while the control group had no obvious variation. The relative contents were 18.54, 46.28, 59.55, 156.76, 162.76, 235.38, and 241.74 before enzyme reaction and reduced to 0, 0, 0, 25.44, 25.78, 69.03, and 70.83, respectively.

Discussion The dose of 3-MCPD was set at 30 mg/kg/day, which is onefifth of the LD50 (20), and has been reported to cause lower body weight gain, increased relative weight of the kidney, and chronic progressive nephropathy and testopathy (21). This dose is sufficiently high to cause metabolic profile differences between treated rats and controls and is also suitable for longterm study. In the present study, these changes were wellreplicated. The value of urine NAG, serum creatinine, and BUN showed elevations in the 3-MCPD-treated group, the variation of which could imply nephrotoxicity. Histopathology showed obvious hydropic degeneration in kidneys and a substantial decrease in spermatozoa and sustentacular cells in seminiferous tubules. All of these results indicated that, with continuous administration for 40 days, we successfully created a rat model of 3-MCPD toxicity. 3-MCPD has general toxicity, including reproductive toxicity (3), genotoxicity (4), carcinogenicity (5), and neurotoxicity (1);

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therefore, it is necessary to monitor the metabolic variation caused by 3-MCPD stimulation, which can be performed by metabonomics. Urine samples of both groups were analyzed by UPLC/Q-TOF/MS with ESI in positive and negative ion modes. Compounds with a molecular weight of 100-1000 were detected, and the data including m/z, retention time, and peak intensity were normalized and exported for statistical analysis. PCA mean score plots in both ion modes revealed that the clustering tendency of metabolites became increasingly obvious with continuous administration of 3-MCPD, and the earliest separation was observed on day 10. Components that played important roles in the separation were picked out according to the parameter variable importance in the projection. We postulated that the identity of the biomarker in this study was galactosylglycerol in negative ion mode. The biomarker was increased significantly in 3-MCPD-treated rats at early time points and at all of the five study periods. Biological functions and metabolic pathways of the biomarker were investigated in databases such as HMDB, Chemspider, and KEGG (Kyoto Encyclopedia of Genes and Genomes). The mechanism of galactosylglycerol elevation in 3-MCPD-treated rats was probably associated with nephrotoxicity and reproductive toxicity, the effect of which might be induced by 3-MCPD toxicity (22). Further research revealed that galactosylglycerol was an early, sensitive biomarker with a high contribution value in clustering and most significant differences on days 10, 20, 30, and 40 (P ) 0.022, 0.007, 0.001, and 0.03, respectively) between the 3-MCPD-treated and the control group. The variation in galactosylglcerol intensity was also coincident with histopathology; both showed early changes on day 10 and more serious variation by continuous treatment. We therefore paid special attention to galactosylglycerol. To avoid contingency of the results, the experiment was duplicated and showed good reproducibility. According to the clinical chemistry results, urine galactosylglycerol intensity had a much earlier increase in the 3-MCPDtreated group than did serum creatinine, BUN concentration, and urine NAG activity. Apparently, the screening tests most widely used to assess the integrity of the kidney, such as serum creatinine and BUN, lack sensitivity (23). Although NAG has been reported to be a useful marker to define effects on the nephron, especially tubular injury (24, 25), which is a lysosomal enzyme with a rich distribution in renal tubular epithelial cells, the sensitivity of urinary NAG was obviously less than galactosylglycerol in the present study. The significant variation of galactosylglycerol occurred at the same time as slight histopathological changes in the kidney and testis and went on relatively in the following time points. The urine galactosylglycerol concentration increased significantly in the 3-MCPD-treated rats on day 10, when the pathological changes, such as hydropic degeneration, were not serious and even reversible. Renal tubule hyperplasia, which was considered to be the most sensitive end point in the long-term

Table 1. Biomarkers in UPLC/MS Positive and Negative Ion Modes retention time (min)

measured m/z ion (Da)

calculated m/z ion (Da)

error of m/z (Da)

7.15 1.42 3.77

310.1029 232.1309 181.0012

positive ion mode 310.0943 0.0086 232.1107 0.0202 181.0535 0.0523

5.03 4.49 5.05 0.32

253.9857 253.0847 254.9907 194.9881

negative ion mode 254.0981 0.1124 253.1002 0.0155 255.0736 0.0829 195.0736 0.0855

elemental composition

postulated identity

C11H19NO9 C10H17NO5 C8H8N2O3

N-acetylneuraminic acid isovalerylglutamic acid nicotinuric acid

C11H15N2O5 C9H18O8 C15H12O4 C10H12O4

nicotinamide riboside galactosylglycerol dihydrodaidzein homoveratric acid

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Figure 3. (A) UPLC/MS negative ion metabolic alterations for galactosylglycerol (A) between controls and 3-MCPD-treated rats at five time points. (B) Urine β-GAL activity comparison between controls and 3-MCPD-treated rats at five time points (mean ( SD, *P < 0.05, and **P < 0.01). (C) UPLC/MS/MS product ion spectra for the ion at m/z 253 (galactosylglycerol).

study of 3-MCPD toxicity, was observed in 3-MCPD-treated rats on day 40, which was much later than the changes in galactosylglycerol. All of these results indicate that urine galactosylglycerol could be an early, sensitive biomarker for evaluation of the adverse effects of 3-MCPD exposure and could vary at the very early period of reversible histopathological injury. The increased levels of galactosylglycerol in urine were likely to have arisen from an effect of 3-MCPD on the activity of β-Gal. β-Gal is a lysosomal enzyme with an abundant distribution in the epididymis and a slightly lower amount in the kidney, which hydrolyzes terminal nonreducing β-Dgalactose residues in β-D-galactosides (22). It has been suggested previously that, in the glycerolipid metabolism pathway, galactosylglycerol is the product of 1,2-diacyl-3β-D-galactosyl-sn-glycerol, which is catalyzed by galactolipase. The following event is hydrolysis by β-Gal, which catalyzes galactosylglycerol and water into galactose and glycerol. It has been shown that the kidney is the target organ of 3-MCPD (26, 27). 3-MCPD also has been reported to inhibit male fertility by decreasing sperm motility, altering sperm morphology, and causing testicular and epididymal damage (3). The continual administration of 3-MCPD to male rats resulted in nephrotoxicity and reproductive toxicity; therefore, the amount of β-Gal distribution in kidney and excreted by the epididymis may have decreased. The depressed activity of β-Gal led to decreased hydrolysis of galactosylglycerol to galactose and glycerol, which could have resulted in elevation of galactosylglycerol concentration. The elevation of urine β-Gal activity could well confirm this hypothesis. An authentic standard of galactosylglycerol was unavailable; thus, it was difficult to make an unequivocal structural determination. β-Gal was added to urine for determination, by which galactosyglycerol can be catalyzed and catabolized. The normal-

ized peak intensity of the compound with a molecular weight of 253 and retention time of 4.49 min was reduced significantly after the enzyme reaction. According to KEGG, of all of the β-Gal-related pathways, the only compound with a molecular weight of approximately 253 that can be degraded by β-Gal is galactosylglycerol, which could confirm the identity of galactosylglycerol. In the present study, the largest relative content reduction of galactosyglycerol in the seven urine samples was from 241.74 to 70.83, which was catabolized by 100 µL of purified β-Gal with an activity of 1144 U/mL. The highest urine β-Gal activity was only 0.036 U/mL, which was much lower than the purified β-Gal. As a result, the relative content of urine galactosyglycerol could hardly be effected by urine β-Gal.

Conclusions The time-dependent metabolic profiles of 3-MCPD-treated rats were found to differ significantly from those of control rats, with a number of markers that contributed to the clustering. Galactosylglycerol was one of those high contribution ratio markers with an early and sensitive variation, which could be used as a biomarker for monitoring 3-MCPD exposure. Future research will study the relationship between exposure to 3-MCPD, galactosylglycerol level, and renal function (e.g., glomerular filtration rate and serum creatinine) in humans. If the relationship is validated, the acceptable daily intake of 3-MCPD in humans and the concentration limits in food could be established, which could reduce the level of exposure to 3-MCPD and the risk of resultant disease. Acknowledgment. This work was supported by the Natural Science Foundation of China (Grant 30471445) and a grant from the Ministry of Health (200902009). We thank the Department of Health Statistics of Harbin Medical University for statistical support and the Pharmacy Department of Heilongjiang University of Chinese Medicine for technical assistance.

NoVel Biomarkers of 3-Chloro-1,2-propanediol Exposure

Note Added after ASAP Publication. This paper was published on the Web on February 17, 2010, with errors in the calculated m/z values in Table 1. Thus, the markers Nacetylneuraminic acid, isovalerylglutamic acid, nicotinuric acid, dihydrodaidzein, nicotinamide riboside, and homoveratric acid were incorrectly identified and have been deleted. The m/z value of galactosylglycerol has been corrected to 253.0923, and the identification of this compound as a biomarker of 3-chloro-1,2propanediol exposure remains valid. The corrected version was reposted on May 12, 2010. Supporting Information Available: Figures detailing body weight and clinical chemistry comparison between controls and 3-MCPD-treated rats (Figure 1), photomicrographs of testis before and after administration (Figure 2), and UPLC/MS data (Figure 3). This material is available free of charge via the Internet at http://pubs.acs.org.

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